PROJECT SUMMARY: In neurons, synaptic vesicles (SV) packaged with neurotransmitter fuse with the plasma membrane to release their content that is sensed across the synaptic cleft. Release is triggered by a local increase in the calcium concentration following depolarization. Release kinetics comprise a synchronous phase (0.1-5 ms after calcium elevation), and a much slower asynchronous phase (~100 ms). How membrane fusion can be triggered so rapidly and how the kinetics are regulated are not well understood. Hormones are released in a similar fashion, with multiple kinetic phases, using some of the same protein machinery, via fusion of hormone containing secretory granules (SG) with the plasma membrane. The initial ~1-3 nm wide connection between the fusing compartments, called the fusion pore, can flicker open-closed in succession before either closing permanently (transient fusion) or dilating fully. There is large variability between cell types (pore open times span ~100 s to 10s of s) and within the same cell (some pores flicker, some dilate abruptly). Pore flickering is modulated by physiological inputs such as stimulation strength, with important consequences about what is released (only small cargo can escape through a small pore), on what time course, and how exocytosis is coupled to endocytosis. Despite the importance of fusion pores in regulating release, very little is understood regarding mechanisms controlling pore nucleation and dynamics. This is mainly due to difficulties in studying fusion pores in reconstituted systems with well-defined protein and membrane components that would allow isolating the role of each. Fusion mediated by exocytic SNARE proteins and their regulators has been reconstituted and studied for the past 20 years. However, methods that can monitor single reconstituted fusion pores with sub- ms resolution have been lacking. During the last cycle, we developed such methods for the first time, and explored mechanisms regulating fusion pores induced by SNAREs alone. In the next cycle, we propose to use those methods to (1) define the role of SNARE-interacting proteins in nucleation and dynamics of fusion pores and the selectivity of small pores. To characterize how the calcium sensors for exocytosis and other essential components of the release machinery contribute to fusion pore properties, we will use electrophysiology, nanodiscs, engineered cells, single-particle fluorescence microscopy, microfabricated devices, and artificial bilayers. We will also characterize selectivity of small fusion pores for ions, which is highly relevant for determining what is released during transient fusion events. We will then (2) dissect mechanisms contributing to kinetics of calcium-triggered exocytosis. The approaches will be augmented to allow rapid (~1 ms) [Ca2+] elevation using microperfusion or ultraviolet flash photolysis. These will enable defining how different sensors and release complexes regulate release kinetics and what determines the high calcium-cooperativity of release. These fundamental studies will advance our understanding of how neurotransmitter and hormone release are regulated, with potential impact on human health in the long term.